This project will use a genetic and biochemical approach to study the molecular mechanism by which mutations are introduced during DNA replication and DNA repair. An in vitro system able to replicate bacteriophage T7 DNA and encapsulate the product into phage heads will be used to produce infective phage in vitro. This system is also able to repair some types of DNA damage. The reactions will be perturbed in ways that might reduce the fidelity of DNA replication and repair, and the phage produced in vitro will be examined for mutations. In vitro mutagenesis will be examined by: altering the pools of DNA precursors, including analogues of normal precursors, and using damaged DNA as a template. The location and base sequence near to the mutation site will be considered. Site directed mutagenesis will be investigated by attempting to recombine mutagen treated T7 DNA restriction fragments into intact T7 genomes in vitro. Also, T7 genomes damaged by ultraviolet radiation or alkylating chemicals will be repaired in vitro, and measurements of mutation induction consequential to DNA repair will be made. Special attention will be given to inducible repair modes that require new protein synthesis for error-free (adaptive response) or error-prone (SOS-response) DNA repair. The intent is to determine the mechanisms by which mutations are introduced during constitutive and inducible modes of DNA repair. In another part of this study heteroduplex T7 DNA will be encapsulated and examined for mismatch repair, and the in vitro DNA replication system will be used in an effort to defect mismatch repair in vitro. To assist in these mutagenesis studies in further improvements in the T7 DNA packaging system are planned. This will involve isolation and study of structures important in T7 bacteriophage assembly and an effort to understand the mechanism of DNA encapsulation. The long term goal of this project is to provide basic knowledge that will allow more meaningful judgments and more accurate extrapolations to be made concerning health hazards caused by genetic damage inflicted by environmental insults.